The present invention relates to a method for the epoxidation of a compound having at least one carbon-carbon double bond, the method involves reacting a compound having at least one carbon-carbon double bond, a solvent, an oxidant, and membrane bound peroxygenase. The present invention also relates to a method for preparing the membrane bound peroxygenase involving grinding seeds containing peroxygenase to produce ground seeds, homogenizing the ground seeds in a buffer to form a slurry, centrifuging the slurry to produce a first supernatant, centrifuging the first supernatant to produce a second supernatant, and filtering said second supernatant through a protein-binding membrane filter to produce membrane bound peroxygenase; optionally the second supernatant is filtered through a hydrophilic membrane filter prior to filtering the second supernatant through the protein-binding membrane filter.
Fats and oils (e.g., soybean oil and the esters of tall oil fatty acids) are renewable materials that are used as feed stocks for the production of industrial materials such as paints, varnishes, emulsifiers, and lubricants. To achieve a formulation with the desired properties, it is usually necessary to chemically modify the fat or oil. For unsaturated fats and oils, one common modification is epoxidation which leads to a material with increased polarity and enhanced reactivity (Carlson, K. D., et al., J. Am. Oil Chem. Soc., 62: 934-939 (1985); Piazza, G. J., Some Recent Advances in Epoxide Synthesis, IN Recent Developments in the Synthesis of Fatty Acid Derivatives, edited by G. Knothe and J. T. P. Derksen, AOCS Press, Champaign, 1999, pp. 182-195)). Epoxidized oils are used as plasticizers and are generated on an industrial scale using peracids (Gan, L. H., et al., J. Am. Oil Chem. Soc., 69: 347-351 (1992)). The latter are generated by reacting formic or acetic acid with hydrogen peroxide in the presence of a strong acid catalyst. A disadvantage of this procedure is that the strong acid catalyzes epoxide ring opening, causes equipment corrosion, and it must be recycled or neutralized before discharge into the environment. Also the peracid intermediate is unstable, and explosive conditions are possible.
The use of enzymes offers the possibility of developing an environmentally benign and more selective epoxidation reaction. One enzyme that might be useful for this purpose is termed peroxygenase. This enzyme catalyzes the heterolytic cleavage of a peroxygen bond and transfers the liberated oxygen to an oxidizable functional group, such as a carbon-carbon double bond, to give an epoxide product. Thus, in the presence of organic hydroperoxide, oleic acid 1 is converted to the 9,10-epoxide 2 by peroxygenase isolated from soybean, broad bean, and oat (FIG. 1) (Hamberg, M., et al., Arch. Biochem. Biophys., 283: 409-416 (1990); Hamberg, M., et al., Plant Physiol., 110: 807-815 (1996); Hamberg, M., et al., Plant. Physiol., 99: 987-995 (1992); Blée, E., et al., J. Biol. Chem., 268:1708-1715 (1993); Blée, E., Phytooxylipins: The Peroxygenase Pathway, IN Lipoxygenase and Lipoxygenase Pathway Enzymes, edited by G. J. Piazza, AOCS Press, Champaign, 1996, pp. 138-161)). Similarly linoleic acid afforded the 9,10- and 12,13-epoxy derivatives. Studies with peroxygenase from soybean and broad bean show that cis-double bonds are the preferred substrates of peroxygenase (Hamberg, M., et al., Plant. Physiol., 99: 987-995 (1992); Blée, E., et al., J. Biol. Chem., 265: 12887-12894 (1990)). Peroxygenase can also catalyze internal epoxidation if the peroxygen group is contained in a molecule with a double bond. Thus when the peroxygenases from soybean and broad bean were presented with the enzymatically-generated hydroperoxide of linoleic acid 3 (13(S)-hydroperoxy-9(Z),11(E)-ocatdecadienoic acid, HPODE), products were 13(S)-hydroxy-9(Z),11(E)-octadecadienoic acid 4 and 9,10-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid 5 (Hamberg, M., et al., Biochem. Biophys., 283: 409-416 (1990); Hamberg, M., et al., Arch. Biochem. Biophys., 283: 409-416 (1990); Blée, E., et al., J. Biol. Chem., 268:1708-1715 (1993); Piazza, G. J., et al., J. Am. Oil Chem. Soc., 76: 551-555 (1999)). Recently it has been demonstrated that a peroxygenase from oat seeds can catalyze the epoxidation of oleic acid using hydrogen peroxide as the oxygen donor (Hamberg, M., et al., Plant Physiol., 110: 807-815 (1996)).
Traditional methods for using an enzyme for synthesis require two separate steps: isolation/purification of the enzyme from its biological source and chemical or physical immobilization. In the study described herein, a simple, rapid method for immobilizing oat seed peroxygenase on filter membranes is described. The activity and reusability of the immobilized peroxygenase preparation also was investigated, and the pH and temperature dependence of epoxidation by membrane-bound peroxygenase was examined to determine optimal reaction conditions.